JP2023151332A - Renal markers and use thereof - Google Patents

Abstract

To provide biomarkers for diagnosing renal functions of cats or dogs, and a diagnostic method using the same.SOLUTION: A method of diagnosing renal functions of cats or dogs is provided, involving diagnosing renal functions based on analysis of metabolites in urine, where the metabolite analysis comprises checking a decrease in the level of at least one substance selected from the group consisting of 2-hydroxyisobutyrate, 2-phenylpropionate, 3-aminoisobutyrate, adenosine, glycine, and trigonelline, or an increase in the level of at least one substance selected from the group consisting of dimethyl sulfone, dimethylamine, methanol, taurine, 2-oxoglutarate, indole-3-acetate, N-phenylacetylglycine, and urocanate.SELECTED DRAWING: None

Description

TECHNICAL FIELD The present invention relates to renal disease markers and their use.

Kidney disease in cats is one of the leading causes of death. Therefore, it is important to accurately diagnose renal function abnormalities using testing methods that can evaluate them at an early stage and to understand the pathological condition. However, there are few reports on research in this field in veterinary clinical practice, and in actual clinical practice, plasma creatinine, SDMA ( Currently, the classification is based on symmetrical dimethylarginine), and when the kidney disease has advanced to a certain stage, breeders often visit the hospital because they are drinking and urinating too much.

Regarding the diagnosis of kidney disease, Patent Documents 1 and 2 describe a method for determining whether an animal is suffering from kidney disease, which method uses β-aminoisobutyric acid (β-aminoisobutyric acid) in a urine sample or blood sample from the animal. A method is described that includes measuring BAIB) and determining renal disease based on the concentration of BAIB in a sample. Further, Patent Document 3 describes a method for analyzing the urine of a subject suspected of having renal failure, in which D-serine and L-serine, D-histidine and L-histidine, and D-asparagine are detected in the urine of the subject. and L-asparagine, D-arginine and L-arginine, D-allo-threonine and L-threonine, D-glutamic acid and L-glutamic acid, D-alanine and L-alanine, D-proline and L- At least one amino acid from the amino acid group consisting of proline, D-valine and L-valine, D-allo-isoleucine and L-isoleucine, D-phenylalanine and L-phenylalanine, D-lysine and L-lysine a step of measuring the concentration of the D-form and L-form pair of the at least one type of amino acid, and calculating a pathological condition index value indicating the composition ratio of the D-form from the concentration of the D-form and L-form pair of the at least one type of amino acid. An analysis method is described that includes the steps of: determining a significant difference in the pathological condition index value, and correlating the significant decrease in the disease state index value with renal failure in the subject.

On the other hand, pharmaceutical or food compositions to be administered to and ingested by subjects with decreased renal function or subjects at risk thereof have been studied. In this regard, Patent Document 4 describes a composition for regulating URAT1 (urate transporter 1) activity, which is characterized by containing taurine as an active ingredient. According to this literature, URAT1 expressed in the kidney is known to be involved in the transport of factors that play important roles in the body, such as uric acid, nicotinic acid, and succinic acid, as well as drugs such as salicylic acid and indomethacin. This study explains that it is suggested that biological functions may be better controlled by regulating its activity. Furthermore, Patent Document 5 discloses that the compound contains threonine, proline, glycine, valine, isoleucine, leucine, tyrosine, phenylalanine, lysine, aspartic acid, serine, glutamic acid, alanine, methionine, tryptophan, histidine, and arginine. Amino acid compositions or amino acid solutions for improving renal dysfunction are described. Further, U.S. Pat. No. 5,020,301 describes an amount of one or more antioxidants that improves kidney function and an amount that is less than the maximum amounts of protein and phosphorus typically recommended for healthy animals of the same species or breed. Compositions suitable for improving renal function in animals are described, containing proteins and/or phosphorus, with preferred antioxidants including β-carotene, selenium, coenzyme Q10 (ubiquinone), luetin, Tocotrienols, soy isoflavones, S-adenosylmethionine, glutathione, taurine, N-acetylcysteine, vitamin E, vitamin C, alpha-lipoic acid and L-carnitine are mentioned.

On the other hand, the present inventors found that docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) was used as an active ingredient for maintaining and protecting renal function, based on the results of experiments conducted on spontaneously hypertensive and stroke-prone rats. A renal function maintenance and protection agent is being developed that is characterized by containing a triglyceride or a mixture of a triglyceride of docosahexaenoic acid (DHA) and a triglyceride of eicosapentaenoic acid (EPA) (Patent Document 7). In addition, a cat renal function protective agent containing an ester of docosahexaenoic acid (DHA) has been developed (Patent Document 8).

International Publication WO2016/176691 (Special Table 2018-515761, Patent No. 6923449) Japanese Patent Application Publication No. 2021-181994 (divisional application of Patent Document 1) Patent No. 5740523 International publication WO2013/018706 Japanese Patent Application Publication No. 2002-275059 Japanese Patent Application Publication No. 2015-7093 JP2020-2125 Publication JP 2022-26979 Publication

IRIS Kidney - Guidelines - IRIS Staging of CKD (http://www.iris-kidney.com/guidelines/staging.html)

Plasma creatinine is known to have a so-called blind area, where factors other than the kidneys, such as gender, age, and muscle mass, affect test data and do not show abnormal values in patients with mild renal impairment. Conversely, if plasma creatinine increases due to muscle mass or exercise, renal function may be underestimated. On the other hand, urea nitrogen has many variable factors such as protein intake and is not specific to renal function.

Urinary protein tests and urine albumin tests are also performed, but urine protein tests may be positive regardless of the presence or absence of renal disease. In addition, although the urine albumin test uses a human urine albumin measurement method, and although there is some homology, the measurement sensitivity is low, and there are no biomarkers with high sensitivity and specificity that can evaluate renal function abnormalities at an early stage. desired.

One of the biomarker search methods is metabolomics, a method that comprehensively measures the metabolome, which is the total number of metabolites. Although it is widely used in preclinical and clinical settings for human drug development, in the veterinary field, there are still only a few examples of metabolomic analysis. This time, we measured urinary metabolites using nuclear magnetic resonance (NMR) in the urine of healthy cats or cats with renal disease, and measured metabolites specific to renal disease and creatinine levels in plasma. I tried to make predictions from things.

The present invention provides the following.
[1] A method for diagnosing renal function in cats or dogs, comprising:
This is a diagnostic method based on the analysis of metabolites in urine.
Analysis of metabolites
2-hydroxyisobutyrate, 2-phenylpropionate, 3-aminoisobutyrate, adenosine, glycine, and trigonelline at least one lowering selected from the group consisting of, or dimethyl sulfone, dimethylamine, methanol, taurine, 2-oxoglutarate, indole-3 - A method of increasing at least one selected from the group consisting of indole-3-acetate, N-phenylacetylglycine, and urocanate.
[2] The method according to 1, wherein the metabolite analysis includes at least one analysis selected from the group consisting of 2-hydroxyisobutyrate, trigonelline, methanol, and urocanate.
[3] The method according to 1 or 2 for diagnosing a cat or dog at stages 1 to 4 of the International Renal Interest Society (IRIS) diagnostic criteria for chronic kidney disease.
[4] A method for predicting a subject's plasma creatinine level, comprising:
This is a prediction method based on the analysis of metabolites in urine.
A method wherein the analysis of metabolites comprises analysis of two or more selected from the group consisting of 2-hydroxyisobutyrate, trigonelline, methanol, urocanate, and lactate.
[5] The prediction method according to 4, wherein the metabolite analysis is an analysis of 2-hydroxyisobutyrate, trigonelline, methanol, urocanate, and lactate.
[6] A method for diagnosing renal function in a cat or dog, comprising:
It is a diagnostic method based on the analysis of metabolites in plasma,
Analysis of metabolites
N,N-Dimethylglycine, Proline, Urea, 5-Hydroxylysine, Asparagine, 5-oxo-2-tetrahydrofurancarboxylic acid (5 -Oxo-2-tetrahydrofurancarboxylic acid), Azelaic acid, Carboxymethyl lysine, Cys, Cystine, Glucaric acid, Hydroxyproline, Methionine sulfoxide ( Methionine sulfoxide, N6-Acetyllysine, Pelargonic acid, Pimelic acid, Pipecolic acid, Stachydrine, Sulfotyrosine, Taurocholic or γ-Glu-Ornithine.
1-Methyladenosine, 2-Aminobutyric acid, 2-Hydroxybutyric acid, 2-Hydroxyoctanoic acid, 3-methoxytyrosine -Methoxytyrosine), 3-Ureidoisobutyric acid, 3-Ureidopropionic acid, 7-Methylguanine, Allantoic acid, Argininosuccinic acid , Ascorbate 2-sulfate, Carnosine, cis-Aconitic acid, Cystathionine, Ethanolamine phosphate, Gluconic acid, Gluconolactone, Gly-Asp, Indole-3-acetic acid, Isethionic acid, Isocitric acid, Isovalerylcarnitine ( Isovalerylcarnitine), N-Acetylaspartic acid, N-Acetylglycine, N-Acetylputrescine, N1-Methylguanosine, N6,N6,N6 - At least one member selected from the group consisting of N6,N6,N6-Trimethyllysine, Nalidixic acid, Phenaceturic acid, Putrescine, Pyridoxal, and Uridine. One way to rise.
[7] A method for diagnosing renal function in a cat or dog, comprising:
It is a diagnostic method based on the analysis of metabolites in plasma,
At least one decrease selected from the group consisting of Proline and γ-Glu-Ornithine, or ADMA, Isethionic acid, 2-amino A method of increasing at least one selected from the group consisting of 2-Aminobutyric acid, 2-Hydroxybutyric acid, and 3-Ureidopropionic acid.
[8] The method according to 6 or 7, wherein the metabolite analysis includes at least one analysis selected from the group consisting of isethionic acid, 2-aminobutyric acid, 2-hydroxybutyric acid, 3-ureidopropionic acid, and proline. Method.
[9] The method according to any one of items 6 to 8 for diagnosing a cat or dog at stage 1 or 2 of the IRIS diagnostic criteria for chronic kidney disease.
[10] A pharmaceutical or food composition containing any one selected from the group consisting of taurine, proline, and γ-glutamyl ornithine, to be ingested by a subject with decreased renal function or a subject at risk thereof.
[11] The composition according to 10, wherein the subject is a cat or a dog.
[12] The composition according to 10 or 11, wherein the subject is a cat or dog in stage 1 or 2 of the IRIS diagnostic criteria for chronic kidney disease.
[13] The composition according to any one of 10 to 12, further comprising at least one selected from the group consisting of docosahexaenoic acid (DHA)-binding lipids and eicosapentaenoic acid (EPA)-binding lipids. .
[14] The composition according to any one of 10 to 13, which is ingested by a subject diagnosed with renal function by the method according to any one of 1 to 9.
[15] From the group consisting of docosahexaenoic acid (DHA)-binding lipids and eicosapentaenoic acid (EPA)-binding lipids, for ingestion by subjects diagnosed with renal function by the method according to any one of 1 to 9. A pharmaceutical or food composition comprising at least one selected from the group consisting of:
[16] The composition according to any one of 13 to 15, which contains at least one selected from the group consisting of DHA-binding lipids and EPA-binding lipids as fish oil.

Prediction of plasma creatinine from urinary metabolites by orthogonal partial least squares regression (full dataset and variable importance for prediction (VIP)) (n=45, 40 metabolites) R2X =0.383, R2Y=0.935 , Q2Y=0.51 Relationship between actual values and predicted values of multiple regression analysis using 5 metabolites Multiple R-squared: 0.7806, Adjusted R-squared: 0.7525, F-statistic: 27.75, p-value:7.14E-12

[Analysis of metabolites in urine]
The present invention relates to a method for diagnosing renal function in cats or dogs, and markers used for the same. The method of the present invention uses metabolites in urine as markers, and diagnoses the renal function of a subject based on the analysis of the markers. Analysis includes detection of the target substance, quantification, and comparison with standard values to determine (decreased or increased). A decrease includes a value lower than that of a healthy group, and an increase includes a value higher than a value of a healthy group.

In one preferred embodiment, the analysis of metabolites includes 2-hydroxyisobutyrate, 2-phenylpropionate, 3-aminoisobutyrate, adenosine ( adenosine), glycine, and trigonelline.

Regarding the present invention, if the substance to be analyzed is expressed as an anion (salt, ester) (represented by replacing -ic acid with -ate), the substance can be measured. Depending on the conditions, it may be measured as an acid. That is, in the present invention, anionic substances listed as targets of analysis may be measured as acids depending on the analysis conditions. For example, 2-hydroxyisobutyrate, 2-phenylpropionate, and 3-aminoisobutyrate are, in order, 2-hydroxyisobutyric acid), 2-phenylpropionic acid, and 3-aminoisobutyric acid. In the present invention, the analysis of 2-hydroxyisobutyrate also includes the analysis of 2-hydroxyisobutyric acid. The same applies to other substances.

In another preferred embodiment, the analysis of metabolites includes dimethyl sulfone, dimethylamine, methanol, taurine, 2-oxoglutarate or 2-oxoglutarate. (2-oxoglutaric acid), indole-3-acetate or indole-3-acetic acid, N-phenylacetylglycine, and urocanate at least one increase selected from the group consisting of:

Specifically, when targeting chronic kidney disease (polycystic kidney disease (PKD) + other chronic kidney diseases (CKD)), metabolite analysis is performed using 2-hydroxyisobutyrate, 2- A decrease in any one selected from the group consisting of phenylpropionate, 3-aminoisobutyrate, adenosine, glycine, and trigonelline, or an increase in any one selected from the group consisting of dimethyl sulfone, dimethylamine, methanol, and taurine. It is preferable.

In addition, when targeting PKD, metabolite analysis is performed to detect a decrease in any one selected from the group consisting of 2-hydroxyisobutyrate, 2-phenylpropionate, 3-aminoisobutyrate, adenosine, glycine, and trigonelline, or 2-oxoglutarate. , dimethyl sulfone, dimethylamine, indole-3-acetate, methanol, N-phenylacetylglycine, taurine, and urocanate.

In particularly preferred embodiments, the metabolite analysis is at least one analysis selected from the group consisting of 2-hydroxyisobutyrate, trigonelline, methanol, and urocanate.

The method of analyzing the target substance is not particularly limited. Those skilled in the art can collect urine using an appropriate method, perform centrifugation if necessary to remove insoluble materials, and add a buffer solution to prepare a sample for measurement. The sample can be analyzed using, for example, nuclear magnetic resonance (NMR), gas chromatography (GC), high performance chromatography (HPLC), mass spectrometry (MS), or time-of-flight mass spectrometry (TOF-MS). I can do it. In addition, if appropriate for a particular substance, analysis can be performed using immunological techniques. The collected urine sample may be subjected to various biochemical tests, such as urine specific gravity, pH, and UPC (urine protein: creatinine), for reference.

[Method to predict plasma creatinine level]
The present invention also relates to a method for predicting plasma creatinine levels in a subject based on analysis of metabolites in urine.

For the purpose of predicting the plasma creatinine value of a subject, the analysis of metabolites in the urine consists of Methanol, 2-Hydroxyisobutyrate, N-Acetylglycine, Tyrosine, indole-3-acetate, Formate, Urocanate, Trigonelline, Lactate. It is preferable to use one selected from the group, and more preferably one selected from the group consisting of 2-Hydroxyisobutyrate, Trigonelline, Methanol, Urocanate, and Lactate.

In order to predict plasma creatinine levels, the present inventor determined that Methanol, 2-Hydroxyisobutyrate and We selected nine metabolites: , N-Acetylglycine, Tyrosine, indole-3-acetate, Formate, Urocanate, Trigonelline, and Lactate, and performed multiple regression analysis using stepwise variable selection (decrease method) using p-values. . As a result, we obtained the following regression equation using five metabolites: Lactate, Methanol, Urocanate, 2-Hydroxyisobutyrate, and Trigonelline.

In the formula, [Lactate], [Methanol], [Urocanate], [2-Hydroxyisobutyrate], and [Trigonelline] represent the concentration of each substance in urine (/Cre). The calculated [Plasma Creatinine] represents the concentration of plasma creatinine (mg/dL).

The method of analyzing the target substance in urine for predicting the plasma creatinine value is not particularly limited. Those skilled in the art can do this as appropriate. An example of an analysis method is the aforementioned nuclear magnetic resonance apparatus (NMR).

Traditionally, CKD grades have been classified based on plasma creatinine levels obtained by collecting blood from subjects, but it is difficult to collect blood frequently. However, according to the present invention, plasma creatinine values can be predicted from urinary metabolites. It is easy for breeders to diagnose, and it is useful for selecting therapeutic foods and comprehensive nutritional foods based on the diagnosis results.

[Analysis of metabolites in plasma]
The present invention also relates to a method of diagnosing a subject's renal function, which is based on analysis of metabolites in plasma. Analysis of metabolites in plasma can be performed as analysis of metabolites in blood or serum. In the following, the case of analyzing metabolites in plasma will be explained as an example.

In a preferred embodiment, for the purpose of diagnosing the kidney function of a subject, the analysis of metabolites in plasma includes N,N-Dimethylglycine, Proline, and Urea. , 5-Hydroxylysine, Asparagine (Asn), 5-Oxo-2-tetrahydrofurancarboxylic acid, Azelaic acid, Carboxymethyllysine, Cys, Cystine, Glucaric acid, Hydroxyproline, Methionine sulfoxide, N6-Acetyllysine, Pelargonic acid, Pimelic acid ( A group consisting of Pimelic acid, Pipecolic acid, Stachydrine, Sulfotyrosine, Taurocholic acid, Terephthalic acid, and γ-Glu-Ornithine. at least one reduction selected from

In another preferred embodiment, the analysis of metabolites in plasma includes 1-Methyladenosine, 2-Aminobutyric acid, 2-Hydroxybutyric acid, 2-Hydroxybutyric acid, 2-Hydroxyoctanoic acid, 3-Methoxytyrosine, 3-Ureidoisobutyric acid, 3-Ureidopropionic acid, 7-Methylguanine ), Allantoic acid, Argininosuccinic acid, Ascorbate 2-sulfate, Carnosine, cis-Aconitic acid, Cystathionine, Phosphorus Ethanolamine phosphate, Gluconic acid, Gluconolactone, Gly-Asp, Indole-3-acetic acid, Isethionic acid acid), Isocitric acid, Isovalerylcarnitine, N-Acetylaspartic acid, N-Acetylglycine, N-Acetylputrescine, N1-Methylguanosine, N6,N6,N6-Trimethyllysine, Nalidixic acid, Phenaceturic acid, Putrescine, Pyridoxal ), uridine (Uridine).

In another preferred embodiment, the analysis of metabolites in plasma is a reduction in at least one selected from the group consisting of Proline and γ-Glu-Ornithine.

In another preferred embodiment, the analysis of metabolites in plasma includes asymmetric dimethyl-L-arginine (ADMA), isethionic acid, 2-Aminobutyric acid, 2-hydroxybutyric acid (2- at least one increase selected from the group consisting of hydroxybutyric acid), and 3-ureidopropionic acid;

In one particularly preferred embodiment, the analysis of metabolites in plasma is performed using at least one analysis selected from the group consisting of isethionic acid, 2-aminobutyric acid, 2-hydroxybutyric acid, 3-ureidopropionic acid, and proline. be.

The method of analyzing the target substance is not particularly limited. Those skilled in the art can collect blood using an appropriate method, add an anticoagulant or a buffer if necessary, perform centrifugation to remove blood cell components, etc., and obtain plasma, which can be used for measurement. As mentioned above, the measurement can be performed using a nuclear magnetic resonance apparatus (NMR), gas chromatography (GC), high performance chromatography (HPLC), mass spectrometry (MS), or time-of-flight mass spectrometry (TOFMS). In addition, if appropriate for a particular substance, analysis can be performed using immunological techniques.

[Pharmaceutical or food composition]
The present invention also relates to pharmaceutical or food compositions for ingestion by subjects with reduced renal function or at risk thereof. Medicines include veterinary medicines, food compositions include pet foods, and pet foods include comprehensive nutritional foods, snacks, therapeutic foods, and other targeted foods (nutritional supplement foods, calorie supplement foods, and animal supplements). .

<Active ingredient>
The active ingredient of the composition is any selected from the group consisting of taurine, proline, γ-glutamyl ornithine, docosahexaenoic acid (DHA) or its conjugated lipid, and eicosapentaenoic acid (EPA) or its conjugated lipid. The number of active ingredients may be one, or two or more.

In one preferred embodiment, the active ingredient is one selected from the group consisting of taurine, proline, and γ-glutamyl ornithine.

In another preferred embodiment, the active ingredient is one selected from the group consisting of DHA-binding lipids and EPA-binding lipids, with DHA-binding lipids being more preferred. When the DHA-binding lipid is a glyceride ester, DHA may be bound to any one of the α-position, β-position, and γ-position of the glyceride. That is, it may be a mono-, di-, or tri-glyceride of DHA, or a mixed glyceride ester of DHA and other fatty acids. In this case, the content of DHA in the glyceride ester is not particularly limited; -95% by mass, 65-90% by mass, etc. The same applies to EPA-bound lipids.

The raw materials for the DHA-binding lipid and the EPA-binding lipid are not particularly limited, and can be obtained from fish oil, marine animal oil, algae-produced lipid, and crustacean-produced lipid. In a preferred embodiment, the composition includes DHA-bound lipids and EPA-bound lipids as fish oils. The type of fish is not particularly limited, but fish from the family Salmonidae, Herringidae, or Codidae are preferred. The Salmonidae family includes the genus Atlantic Salmon, the genus Atlantic Salmon, the genus Char, and the genus Itu, but fish of the genus Atlantic Salmon or the genus Atlantic Salmon are more preferable. Examples of fish in the Atlantic salmon genus include chum salmon, coho salmon (coho salmon, silver salmon), pink salmon, cherry salmon, yamame, formosan trout, satsuki trout, amago, biwa trout (ameno fish), rainbow trout (steelhead), and masunosuke (masu salmon). King salmon), sockeye salmon, Himemasu, and Kunimasu. Examples of fish belonging to the Atlantic salmon genus include Atlantic salmon and brown trout. The herring family includes the subfamily Herring, the subfamily Herring, the subfamily Ehiravinae, and the subfamily Ehiravinae, but fish of the subfamily Herring and the genus Herring are preferable. Examples of fish of the genus Herring include herring and Atlantic herring. The cod family includes the genera Gadus, Micromesistius, Gadiculus, Trisopterus, Microgadus, Eleginus komai, Merlangius, Melanogrammus, Pollachius, Boreogadus, Arctogadus, but Codaceae Preferably, it is a fish of the genus. Examples of fish in the genus Cod include pollack, cod, Atlantic cod, and Greenland cod. Fish eggs may be natural or farmed.

Preferably, the fish oil is derived from fish eggs. Examples of fish eggs include sujiko, salmon roe, herring roe, and sukeko.

<Application>
The composition can be used for the treatment of renal disease. It can also be used to improve renal function in a subject. Treatment includes reducing the risk of developing, preventing, treating, and inhibiting or halting progression. Treatment includes symptomatic treatment and radical treatment. Kidney diseases include chronic kidney disease and polycystic kidney disease.

The composition can be used to be ingested by a subject diagnosed with decreased renal function by the above-described diagnostic method.

The composition can also be used to reduce the amount of Isethionic acid, 2-Aminobutyric acid, 2-Hydroxybutyric acid, and 3-Ureidopropionic acid in plasma. It can be used to improve the condition of a subject whose condition is low in at least one selected from the group consisting of:

<Dose, content>
The amount of the composition of the present invention to be ingested may be any amount that achieves the desired effect. The intake amount can be appropriately set in consideration of various factors such as the age, weight, and symptoms of the subject.

The content of the active ingredient in the composition can be appropriately determined depending on the form of the composition. For example, if the active ingredient is one of the group consisting of taurine, proline, and γ-glutamyl ornithine, the content of the active ingredient per composition (if there are multiple active ingredients, the total amount of the multiple ingredients) ) can be 0.1% or more, preferably 0.3% or more, for example, 0.6% or more, 1.1% or more, based on dry matter. Regardless of the lower limit, the upper limit of the active ingredient can be 10% or less, 9% or less, 8% or less, and 7% or less on a dry matter basis. It can be set to 6% or less. In the present invention, % means % by mass unless otherwise specified.

When the active ingredient is one of the group consisting of taurine, proline, and γ-glutamylornithine, the daily intake of the composition is the amount of the active ingredient (or, if there is more than one active ingredient, the amount of the multiple ingredients). 10 mg or more, preferably 30 mg or more, more preferably 60 mg or more, and even more preferably 100 mg or more. The upper limit of the active ingredient per day may be 10 g or less, 5 g or less, 1 g or less, and 500 mg, regardless of the lower limit. The amount may be 400 mg or less, 300 mg or less, or 200 mg or less.

The intake may be once a day or multiple times a day, for example 2 to 10 times. The amount of active ingredient ingested per time can be, for example, 1 mg or more, preferably 3 mg or more, more preferably 6 mg or more, and even more preferably 11 mg or more. The upper limit of the active ingredient per serving can be 10 g or less, can be 10 g or less, can be 5 g or less, and can be 1 g or less, regardless of the lower limit value. The amount may be 500 mg or less, may be 400 mg or less, may be 300 mg or less, or may be 200 mg or less.

When the active ingredient is a DHA-binding lipid, the amount of DHA can be 25 mg/kg or more, preferably 50 mg/kg or more, and preferably 100 mg/kg or more. More preferably, the amount is 200 mg/kg or more. The upper limit of the active ingredient per day can be 5000 mg/kg or less, 2500 mg/kg or less, and 2000 mg or less, regardless of the lower limit. The amount can be 1000 mg/kg or less, 750 mg/kg or less, 500 mg/kg or less, or 300 mg/kg or less.

<Other ingredients and additives>
The composition may contain other nutritional and functional ingredients that are acceptable as pharmaceuticals or foods. Examples of such ingredients are lipids (e.g., milk fat, vegetable oils, medium-chain fatty acid-containing fats and oils), proteins, amino acids (e.g., lysine, arginine, glycine, alanine, glutamic acid, leucine, isoleucine, valine), sugars. vitamins (e.g. vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, vitamin D, vitamin E, vitamin K, biotin, folic acid, pantothenic acid and nicotinic acids), electrolytes (e.g. sodium, potassium, calcium, magnesium), minerals (e.g. copper, zinc, iron, cobalt, manganese), dietary fiber, antibiotics, etc.

Furthermore, the composition may further contain additives that are acceptable as pharmaceuticals or foods. Examples of such additives are inert carriers (solid or liquid carriers), excipients, surfactants, binders, disintegrants, lubricants, solubilizing agents, suspending agents, coating agents, colorants. agents, preservatives, buffers, pH adjusters, emulsifiers, stabilizers, sweeteners, antioxidants, flavors, and acidulants.

<Dosage form/form>
The composition may be prepared in any form such as solid, liquid, mixture, suspension, powder, granule, paste, jelly, gel, capsule, etc. It can also be in the form of granules, powder, paste, concentrated liquid, etc., for administration by mixing with drinks or foods. In addition, it can be made into any dosage form suitable for oral administration, such as solid preparations such as tablets, granules, powders, pills, and capsules, liquid preparations such as solutions, suspensions, and syrups, gels, and aerosols. be able to. Further, it can be made into any form such as dry, soft dry, semi-moist, wet, canned, aluminum tray, retort pouch, etc.

<Manufacturing method, display, etc.>
In the production of the composition, the step of blending the active ingredients can be selected as appropriate. There are no particular restrictions on the step of compounding as long as the properties of the active ingredients are not significantly impaired. For example, the active ingredient can be blended with the raw materials. Alternatively, the active ingredient can be added at the final stage of production to produce a composition containing the active ingredient.

The composition of the present invention can be labeled with its purpose of use (application), and can be labeled with a recommendation for administration to a specific subject. Labeling can be direct or indirect; examples of direct labeling are on tangible objects such as the product itself, packages, containers, labels, tags, etc.; examples of indirect labeling are: This includes advertising and promotional activities in any place or by any means, such as websites, stores, pamphlets, exhibitions, seminars such as media seminars, books, newspapers, magazines, television, radio, mail, e-mail, voice, etc.

[others]
In the context of the present invention, unless otherwise specified, the term subject includes cats and dogs, preferably cats. The breeds of cats and dogs are not limited. Targets include healthy individuals at risk of kidney disease. The present invention is suitable for use in cats or dogs in stages 1, 2, 3, or 4 of the IRIS diagnostic criteria for chronic kidney disease, and optionally in cats or dogs in stages 1 or 2. It is suitable for

When referring to amino acids in the present invention, they may be in the L-form or in the D-form, but unless otherwise specified, the L-form is preferred.

Regarding the present invention, the term "ingestion" is used not only to mean ingesting a food composition to a subject, but also to mean administering a pharmaceutical composition to a subject, and "administration" refers to administering a pharmaceutical composition to a subject. It is also used to refer to food compositions being ingested by a subject.

[Analysis of cat urine]
I. Materials and methods
I-1. Subjects Healthy cats diagnosed by a veterinarian specializing in nephrology: n=10, polycystic kidney disease (PKD) cats: n=19, other chronic kidney disease (CKD) Cats: n=13, cats with other diseases: n=3 were tested. The PKD group and CKD group were combined into chronic kidney disease (CKD+PKD) cats: n=32. In addition, healthy cats No. 7 and 9 both exhibited glomerular filtration rates within the reference range and were individuals with large muscle mass.

I-2. Sample Preparation Blood was collected using a vacuum blood collection tube containing EDTA-2Na, and centrifuged (2,000 x g, 4°C, 10 minutes) to obtain plasma. The obtained plasma was subjected to biochemical tests (creatinine).
Urine was collected by cystocentesis, and biochemical tests (urine specific gravity, pH, UPC (urine protein: creatinine)) were measured. A portion of the sample was stored frozen at -80°C until NMR measurement. Cryopreserved urine samples were thawed at 0 to 4°C and centrifuged (2,000 × g, 4°C, 10 minutes). The resulting supernatant (200 μL) was supplemented with 0.2 M phosphate buffer (adjusted to pH 7.4 with sodium dihydrogen phosphate and disodium hydrogen phosphate, 10% (v/v) heavy water, and 3 mM as an internal standard). -Add sodium trimethylsilyl[2,2,3,3,-2 d4]propionate (TSP) (final concentration: 1 mM) (400 μL), mix, and centrifuge (2,000 × g, 4 °C). , for 10 minutes). The obtained supernatant (550 μL) was transferred to an NMR tube and used for measurement.

I-3. NMR measurement JNM-ECZ (500 MHz, JEOL Ltd., Akishima, Tokyo) was used as a nuclear magnetic resonance apparatus (NMR). The measurement conditions are as follows. Measurement temperature: 298 K, observation center: 4.665 ppm, measurement range: 15 ppm, data points: 32,768, number of integrations: 128, delay time: 5 seconds/scan, pulse flip angle: 90°. Eliminated by pre-saturation method.

I-4. Analysis Each measured ^1H -NMR spectrum was analyzed using NMR Suite (ver. 7.4) (Chenomx, Toronto, Canada) to estimate the urinary metabolites and calculate the concentration based on the internal standard sample (TSP). and determined the metabolite concentration. In addition, since the sample was spot urine obtained by cystocentesis, a value corrected by urinary creatinine was used.

I-5. Statistical analysis For comparisons between groups, an unpaired Student's t-test was performed. Orthogonal partial least square regression (OPLSR) was analyzed using R (ver. 3.6.1) and RStudio (ver. 1.1.463). In addition, as pre-processing for analysis, normalization processing was performed in which the average value was converted to 0 and the variance was 1. In addition, the OPLSR model formula was verified using leave-one-out. Multiple regression analysis was performed using R (ver.3.4.1) and EZR (ver.1.37).

II. Results and discussion
II-1. Background information on the test cats Table 1 provides background information regarding the renal function of the test cats.

By ¹ H-NMR, 70 metabolites were estimated as metabolites in cat urine. Among them, 40 metabolites with high identification confidence (Tables 2 and 4) were adopted for analysis. Table 2 shows a comparison of metabolites between healthy cats (n = 10) and cats with chronic kidney disease (CKD + PKD) (n = 32), and Table 3 lists metabolites that vary significantly compared to healthy cats. . Similarly, for comparison between healthy cats and PKD cats, Table 4 and significantly variable metabolites are listed in Table 5.

II-2. Urinary metabolites that distinguish between healthy cats and cats with kidney disease In cats with chronic kidney disease (CKD+PKD), 2-hydroxyisobutyrate, Six metabolites were found: 2-phenylpropionate, 3-aminoisobutyrate, adenosine, glycine, and trigonelline, and four metabolites were found to be excreted significantly in the urine: dimethyl sulfone, dimethylamine, methanol, and taurine.

Furthermore, in PKD, six metabolites, 2-hydroxyisobutyrate, 2-phenylpropionate, 3-aminoisobutyrate, adenosine, glycine, and trigonelline, were significantly lower, while 2-oxoglutarate, dimethyl sulfone, dimethylamine, indole-3-acetate, methanol , N-phenylacetylglycine, taurine, and urocanate were found to be significantly excreted.

In previous studies in humans and rodents (Non-patent Document 2), tryptophan, valine, 3-methyl histidine, glutamine, glycine, homocysteine, phenylalanine, trigonelline were used in CKD, and allantoin, 2-oxoglutaric were used in PKD. Significant changes have been reported for acid, citrate, hippuric acid, malic acid, uric acid, fumaric acid, glutamic acid, glutamine, and hypoxanthine.

Comparing these, trigonelline was consistent across species in cats, while glycine and 2-oxoglutarate showed differences in behavior. Among the metabolites found in this study, taurine was excreted 10 times more in cats with chronic kidney disease (PKD and CKD+PKD) than in healthy cats.

II-3. Model formula for predicting renal function from urinary metabolites Next, we attempted to predict plasma creatinine levels from urinary metabolites. At present, CKD grades are classified based on plasma creatinine levels, but blood sampling from cats is difficult, as they tend to run wild. Therefore, if predictions can be made based on urinary metabolites, it can be expected to lead to self-medication by breeders, and it will also be possible to select therapeutic foods and comprehensive nutritional foods that suit the needs.

Using data from all 45 cats listed in Table 1, we attempted to predict plasma creatinine values from urinary metabolites, that is, construct a regression model, for all cats from healthy to renal disease.

Regression analysis is a method for predicting and explaining response variable Y using explanatory variable X. One type of regression analysis, PLS regression analysis, can reduce the number of variables by determining latent variables with reduced data, without using explanatory variables as they are. For example, in the case of multiple regression analysis, if there is a strong correlation between explanatory variables, there is a high possibility that the variables will influence each other, such as canceling out the predicted part of one variable by another variable, so the reliability It is not possible to create a highly predictive model. This phenomenon is called multicollinearity, and by using latent variables in PLS regression analysis, it is possible to reduce the number of variables used in regression, so the risk of multicollinearity can be reduced. Therefore, it is possible to create a good prediction model using more explanatory variables, which is useful for analyzing multivariate correlations (Non-Patent Document 3).

This time, we constructed a prediction model formula using OPLS, which converts the statistical space into an orthogonal coordinate system that is easy to understand visually. In order to objectively evaluate the quality of the predictive model, we used the R2Y value, which is an indicator of model linearity, and the Q2Y value, which is an indicator of predictive power. The closer the R2Y value and Q2Y value are to 1, the better the prediction model. Specifically, if the R2Y value is 0.65 or more, approximate quantitative prediction is possible, and if the Q2Y value is 0.5 or more, it is considered a good prediction model. (Non-patent Document 4).

Furthermore, by using the VIP value, it is possible to know the contribution of each explanatory variable to the predictive performance of the predictive model. The VIP value is calculated for each explanatory variable, and the larger the value, the greater the contribution to the predictive performance of the predictive model. Therefore, by using the VIP value as an index, we can expect to estimate urinary metabolite components that are considered important for plasma creatinine levels.

In the model (Figure 1) using all data (45 samples, 40 metabolites), a relatively good model could be constructed with R2Y=0.935 and Q2Y=0.51. In the graph, the vertical axis (actual) shows the actual plasma creatinine value, and the horizontal axis (predicted) shows the plasma creatinine value estimated from urinary metabolites, which are plotted for each sample. If the predicted value and the measured value match, it will be on the y=x line.

In view of practical application, a prediction model that has fewer variables and is easier to calculate is required. Among the 40 metabolites in the previous model, 9 metabolites with high VIP values maintain the predictive ability of OPLS: Methanol, 2-Hydroxyisobutyrate, N-Acetylglycine, Tyrosine, indole-3-acetate, Formate, Urocanate, Trigonelline, and Lactate. I chose something. Furthermore, as a result of multiple regression analysis using stepwise variable selection (decreasing method) using p values (FIG. 2 and Table 7), five metabolites remained in the end, and the following regression formula was obtained.

It can be seen that the square of the multiple correlation coefficient, which is the correlation between the predicted value and the actual value, is R2=0.7806, which explains about 78% of the plasma creatinine fluctuation. As an evaluation of the entire model, the result of the F test was P<0.001, which showed significant validity. In addition, the variance inflation factor (VIF) value was less than 5 for each variable, and it was judged that the possibility of multicollinearity was low. The above results indicate that a good model formula for predicting plasma creatinine from five urinary metabolites was constructed.

Therefore, by targeting these metabolites, renal function can be predicted using urine collected at home using an NMR or mass spectrometer, test strips that show a specific color reaction, pet sheets, etc. It becomes possible to predict renal function without drawing blood.

III. DHA administration test
III-1. Administration of DHA-rich fish oil to healthy cats and PKD cats Five healthy cats and five PKD cats were given DHA-rich fish oil (DHA-RS: manufactured by Maruha Nichiro) directly or commercially available pet food before evening feeding. (in paste form) and administered 0.45 mL/kg (250 mg/kg as DHA) for 28 days. Three of the PKD cats were treated with a vasopressin receptor antagonist at 3-4 mg/kg. During the intake period, they were given free access to water, and healthy cats were fed 25 g/head of general nutritional food in the morning and evening, and cats with PKD were fed 20 g/head of therapeutic food for renal disease. Before the start of intake and 28 days after intake, general physical examination findings (weight, BCS, TPR, CRT, tent test), blood tests (CBC, blood smear, biochemical tests, a1AG, SDMA), urine tests (urine specific gravity, urine stick, urinary sediment, UPC, FE, urinary NAG index).

General hematology tests and blood biochemistry tests showed normal values. Blood SDMA (symmetrical dimethylarginine), UPC (urine protein: creatinine), and proximal tubular damage marker (urinary NAG index), which are indicators of renal function, decreased in all cases without exception, and as a whole, before and after administration. A significant decrease was observed in (Table 8).

III-2. Taurine in urine 0 for sample preparation, ^1H -NMR measurement, and estimation and quantification of urinary metabolites [analysis of cat urine]
See I. Materials and Methods. Quantitative data were corrected with creatinine. For statistical analysis, R (ver. 3.4.1) and EZR (ver. 1.37) were used, and repeated-measures ANOVA and Tukey-Kramer multiple comparison tests were performed.

As a result of a repeated measures analysis of variance at two levels of test animals (healthy and PKD) and DHA administration (at the start of intake and 28 days after intake), the interaction was significant, so multiple comparisons of all groups were performed, and it was found that urinary Taurine showed a significantly higher level in PKD cats than in healthy cats before the start of intake, but it decreased to the same level as in healthy cats after DHA administration (Table 9). Since taurine is also an essential amino acid for cats, we inferred that it would be effective to create feed enriched with taurine, in addition to the renal protective effect of DHA.

[Metabolomic analysis of cat plasma by CE-TOFMS]
I. Materials and methods
I-1. Subjects Healthy cats: n=5, polycystic kidney disease (PKD) cats: n=5.

I-2. Sample Preparation Blood was collected using a vacuum blood collection tube containing EDTA-2Na, and centrifuged (2,000 x g, 4°C, 10 minutes) to obtain plasma. 30 μL of cat plasma was added to 120 μL of methanol solution prepared so that the concentration of the internal standard substance was 20 μM, and the mixture was stirred. 90 μL of Milli-Q water was added to this, stirred, and transferred to an ultrafiltration tube (Ultrafree MC PLHCC, HMT, centrifugal filter unit 5 kDa). This was centrifuged (9,100 x g, 4°C, 120 minutes) and subjected to ultrafiltration. The filtrate was dried and dissolved in Milli-Q water again for measurement.

I-3. CE-TOFMS measurement In this test, measurements in cation mode and anion mode were performed under the conditions shown below.

Cationic metabolite (cation mode) device
Agilent CE-TOFMS system (Agilent Technologies) Unit 6
Capillary: Fused silica capillary id 50 μm × 80 cm
Measurement condition
Run buffer: Cation Buffer Solution (p/n : H3301-1001)
Rinse buffer: Cation Buffer Solution (p/n : H3301-1001)
Sample injection: Pressure injection 50 mbar, 10 seconds
CE voltage: Positive, 30 kV
MS ionization: ESI Positive
MS capillary voltage: 4,000V
MS scan range: m/z 50-1,000
Sheath liquid: HMT Sheath Liquid (p/n : H3301-1020)

Anionic metabolite (anion mode) device
Agilent CE-TOFMS system (Agilent Technologies) No. 14 Capillary: Fused silica capillary id 50 μm × 80 cm
Measurement condition
Run buffer: Anion Buffer Solution (p/n : I3302-1023)
Rinse buffer: Anion Buffer Solution (p/n : I3302-1023)
Sample injection: Pressure injection 50 mbar, 10 seconds
CE voltage: Positive, 30 kV
MS ionization: ESI Negative
MS capillary voltage: 3,500V
MS scan range: m/z 50-1,000
Sheath liquid: HMT Sheath Liquid (p/n : H3301-1020)

I-4. Analysis
For peaks detected by CE-TOFMS, peaks with a signal/noise (S/N) ratio of 3 or more are automatically extracted using automatic integration software MasterHands ver.2.19.0.2 (developed by Keio University), and the mass Charge ratio (m/z), peak area value, and migration time (MT) were obtained. Metabolites were estimated based on the m/z and MT values for the detected peaks, and relative area values were calculated based on the internal standard sample. For comparisons between groups, an unpaired Student's t-test was performed.

II. Results
II-1. Background information on the test cat Table 10 provides background information regarding the renal function of the test cat.

II-2. Plasma metabolites that distinguish between healthy cats and cats with kidney disease (relative area values)
Metabolome analysis was performed on cat plasma using CE-TOFMS. A candidate compound was estimated at peak 248 (148 cations, 100 anions) from the m/z and MT values of substances registered in the HMT metabolite library and Known-Unknown library (Human Metabolome Technologies, Yamagata). Among the 248 estimated metabolites, 1-Methyladenosine, 2-Aminobutyric acid, 2-Hydroxybutyric acid, 2-Hydroxyoctanoic acid, 3-Methoxytyrosine, 3-Ureidoisobutyric were found to be significantly higher in PKD cats than in healthy cats. acid, 3-Ureidopropionic acid, 7-Methylguanine, ADMA, Allantoic acid, Argininosuccinic acid, Ascorbate 2-sulfate, Carnosine, cis-Aconitic acid, Citrulline, Cystathionine, Ethanolamine phosphate, Gluconic acid, Gluconolactone, Gly-Asp, Indole-3 -acetic acid, Isethionic acid, Isocitric acid, Isovalerylcarnitine, Lys, N-Acetylaspartic acid, N-Acetylglycine, N-Acetylputrescine, N1-Methylguanosine, N6,N6,N6-Trimethyllysine, Nalidixic acid, Phenaceturic acid, Putrescine, Pyridoxal, SDMA , Threonic acid, and Uridine (Table 11), and the metabolites that showed significantly low values were 5-Hydroxylysine, 5-Oxo-2-tetrahydrofurancarboxylic acid, Asn, Azelaic acid, Carboxymethyllysine, Cys, Cystine. , Glucaric acid, Hydroxyproline, Methionine sulfoxide, N,N-Dimethylglycine, N6-Acetyllysine, Ornithine, Pelargonic acid, Pimelic acid, Pipecolic acid, Pro, Stachydrine, Sulfotyrosine, Taurocholic acid, Terephthalic acid, Thr, Trigonelline, Urea, γ- 25 metabolites of Glu-Ornithine were observed (Table 12).

Previous studies in humans and rodents have shown that ADMA, Citrulline, Lysine, N,N-Dimethylglycine, Proline, SDMA, Threonic acid, and Urea are increased and Ornithine, Threonine, and Trigonelline are decreased in CKD. reported (Table 13). Comparing these, in cats, the behavior of ADMA, Citrulline, Lysine, Ornithine, SDMA, Threonine, Threonic acid, Trigonelline was consistent across species, while N,N-Dimethylglycine, Proline, Urea observed a difference in behavior.

III. DHA administration test
III-1. Administration of DHA-rich fish oil to healthy cats and PKD cats Five healthy cats and five PKD cats were given DHA-rich fish oil (DHA-RS: manufactured by Maruha Nichiro) directly or commercially available pet food before evening feeding. (in paste form) and administered 0.45 mL/kg (250 mg/kg as DHA) for 28 days. Three of the PKD cats were treated with a vasopressin receptor antagonist at 3-4 mg/kg. During the intake period, they were allowed to drink water ad libitum, and in the morning and evening, healthy cats were fed 25 g/head of general nutritional food, and cats with PKD were fed 20 g/head of therapeutic food for renal disease. Before the start of intake and 28 days after intake, general physical examination findings (weight, BCS, TPR, CRT, tent test), blood tests (CBC, blood smear, biochemical tests, a1AG, SDMA), urine tests (urine specific gravity, urine stick, urinary sediment, UPC, FE, urinary NAG index).

General hematology tests and blood biochemistry tests showed normal values.
Blood SDMA (symmetrical dimethylarginine), UPC (urine protein: creatinine), and proximal tubular damage marker (urinary NAG index), which are indicators of renal function, decreased in all cases without exception, and as a whole, before and after administration. A significant decrease was observed in (Table 14).

III-2. Metabolites in plasma (relative area values)
0 for sample preparation methods, CE-TOFMS measurements, and estimation and semi-quantification of plasma metabolites [analysis of cat urine]
See I. Materials and Methods. For statistical analysis, R (ver. 3.4.1) and EZR (ver. 1.37) were used, and repeated-measures ANOVA and Tukey-Kramer multiple comparison tests were performed.

As a result of repeated measures analysis of variance at two levels for test animals (healthy and PKD) and DHA administration (at the start of intake and 28 days after intake), metabolites for which interaction was significant were extracted and multiple comparisons were made for all groups. As a result, plasma ADMA, Isethionic acid, SDMA, 2-Aminobutyric acid, 2-Hydroxybutyric acid, and 3-Ureidopropionic acid were significantly higher in PKD cats than in healthy cats before the start of intake; This decreased to a level equivalent to that of healthy cats (Table 15). Conversely, plasma Proline and γ-Glu-Ornithine were significantly lower in PKD cats than in healthy cats before the start of intake, but increased to levels similar to those in healthy cats after DHA administration (Table 16) . In addition to SDMA, which is a conventional indicator of renal function, the above seven metabolites are expected to serve as new indicators of renal function. It was also inferred that it would be effective to create a feed enriched with Proline and γ-Glu-Ornithine.

IV. Results (quantitative values)
IV-1. Plasma metabolites that distinguish between healthy cats and cats with kidney disease (relative area values)
Quantitative analysis was performed on target metabolic compounds. The calibration curve used the peak area corrected with the internal standard substance, and the concentration was calculated for each substance using a one-point calibration of 100 μM (internal standard substance 200 μM).

Quantitative values were calculated for the peaks of 153 detected substances (94 cations, 59 anions) among the target metabolic compounds. Among the estimated 153 metabolites, 1-Methyladenosine, 2-Aminobutyric acid, 2-Hydroxybutyric acid, 3-Ureidopropionic acid, 7-Methylguanine, Allantoic acid were found to be significantly higher in PKD cats than in healthy cats. , Ascorbate 2-sulfate, Carnosine, cis-Aconitic acid, Citrulline, Cystathionine, Ethanolamine phosphate, Gluconic acid, Gly-Asp, Indole-3-acetic acid, Isethionic acid, Isocitric acid, Lys, N-Acetylaspartic acid, N-Acetylglycine , N-Acetylputrescine, Phenaceturic acid, Putrescine, Pyridoxal, Threonic acid, Uridine (Table 16), and the metabolites that showed significantly lower values were 5-Hydroxylysine, Asn, Azelaic acid, Cys, Cystine. , Hydroxyproline, Methionine sulfoxide, N,N-Dimethylglycine, N6-Acetyllysine, Ornithine, Pelargonic acid, Pimelic acid, Pipecolic acid, Pro, Thr, Trigonelline, and Urea (Table 17).

IV-2. DHA administration test (quantitative value)
As a result of repeated measures analysis of variance at two levels for test animals (healthy and PKD) and DHA administration (at the start of intake and 28 days after intake), metabolites for which interaction was significant were extracted and multiple comparisons were made for all groups. However, plasma Isethionic acid, 2-Aminobutyric acid, 2-Hydroxybutyric acid, and 3-Ureidopropionic acid were significantly higher in PKD cats than in healthy cats before ingestion, but with DHA administration, the levels were equal to those in healthy cats. level (Table 18). On the contrary, plasma Proline showed a significantly lower value in PKD cats than in healthy cats before the start of intake, but increased to the same level as in healthy cats by DHA administration (Table 18). In addition to SDMA, which is a conventional indicator of renal function, the above four metabolites can be expected as new indicators of renal function. We also inferred that creating feed enriched with Proline would be effective.

[Production example: Comprehensive nutritional food for cats]
Tuna 10.00g
Soy protein 1.00g
Fish extract 1.00g
Yeast extract 0.30g
Vegetable oil 1.30g
Fish oil 1.00g
Starch 0.50g
Thickener 0.10g
Vitamins/minerals 0.70g
Amino acids (taurine) 0.03g
Water 24.07g
Total 40.0g

The mixture with the above composition is filled into a container, sterilized by retort, and a comprehensive nutritional food for cats containing 0.3% taurine on a dry matter basis is produced.

[Literatures cited in the Examples section]
Non-patent document 2: Abbiss H, Maker GL, Trengove RD. Metabolomics Approaches for the Diagnosis and Understanding of Kidney Diseases. Metabolites. 2019 Feb 14;9(2):34. doi: 10.3390/metabo9020034. PMID: 30769897; PMCID: PMC6410198.
Non-patent document 3: Jonsson, P., Gullberg, J., Nordstrom, A., Kusano, M., Kowalczyk, M., Sjostrom, M., Moritz, T.: A strategy for identifying differences in large series of metabolomics. samples analyzed by GC/MS, Anal. Chem., 2004, 76, 1738-1745.
Non-patent document 4: Eriksson, L. and Johansson, E.: Multi- and megavariate data analysis principles and applications, p. 43-70, 94-97, 105-107, 489-491, Umetrics AB, Umea (2001) .

Claims (16)

A method for diagnosing renal function in a cat or dog, comprising:
This is a diagnostic method based on the analysis of metabolites in urine.
Analysis of metabolites
2-hydroxyisobutyrate, 2-phenylpropionate, 3-aminoisobutyrate, adenosine, glycine, and trigonelline at least one lowering selected from the group consisting of, or dimethyl sulfone, dimethylamine, methanol, taurine, 2-oxoglutarate, indole-3 - A method of increasing at least one selected from the group consisting of indole-3-acetate, N-phenylacetyl glycine, and urocanate. 2. The method of claim 1, wherein the metabolite analysis comprises at least one analysis selected from the group consisting of 2-hydroxyisobutyrate, trigonelline, methanol, and urocanate. 3. The method of claim 1 or 2 for the diagnosis of cats or dogs in stages 1 to 4 of the International Renal Interest Society (IRIS) diagnostic criteria for chronic kidney disease. A method for predicting a plasma creatinine level in a subject, the method comprising:
This is a prediction method based on the analysis of metabolites in urine.
A method wherein the analysis of metabolites comprises analysis of two or more selected from the group consisting of 2-hydroxyisobutyrate, trigonelline, methanol, urocanate, and lactate. 5. The prediction method according to claim 4, wherein the metabolite analysis is an analysis of 2-hydroxyisobutyrate, trigonelline, methanol, urocanate, and lactate. A method for diagnosing renal function in a cat or dog, comprising:
It is a diagnostic method based on the analysis of metabolites in plasma,
Analysis of metabolites
N,N-Dimethylglycine, Proline, Urea, 5-Hydroxylysine, Asparagine, 5-oxo-2-tetrahydrofurancarboxylic acid (5 -Oxo-2-tetrahydrofurancarboxylic acid), Azelaic acid, Carboxymethyl lysine, Cys, Cystine, Glucaric acid, Hydroxyproline, Methionine sulfoxide ( Methionine sulfoxide, N6-Acetyllysine, Pelargonic acid, Pimelic acid, Pipecolic acid, Stachydrine, Sulfotyrosine, Taurocholic or γ-Glu-Ornithine.
1-Methyladenosine, 2-Aminobutyric acid, 2-Hydroxybutyric acid, 2-Hydroxyoctanoic acid, 3-methoxytyrosine -Methoxytyrosine), 3-Ureidoisobutyric acid, 3-Ureidopropionic acid, 7-Methylguanine, Allantoic acid, Argininosuccinic acid , Ascorbate 2-sulfate, Carnosine, cis-Aconitic acid, Cystathionine, Ethanolamine phosphate, Gluconic acid, Gluconolactone, Gly-Asp, Indole-3-acetic acid, Isethionic acid, Isocitric acid, Isovalerylcarnitine ( Isovalerylcarnitine), N-Acetylaspartic acid, N-Acetylglycine, N-Acetylputrescine, N1-Methylguanosine, N6,N6,N6 - At least one member selected from the group consisting of N6,N6,N6-Trimethyllysine, Nalidixic acid, Phenaceturic acid, Putrescine, Pyridoxal, and Uridine. One way to rise. A method for diagnosing renal function in a cat or dog, comprising:
It is a diagnostic method based on the analysis of metabolites in plasma,
At least one decrease selected from the group consisting of Proline and γ-Glu-Ornithine, or ADMA, Isethionic acid, 2-amino A method of increasing at least one selected from the group consisting of 2-Aminobutyric acid, 2-Hydroxybutyric acid, and 3-Ureidopropionic acid. The method according to claim 6 or 7, wherein the metabolite analysis includes at least one analysis selected from the group consisting of isethionic acid, 2-aminobutyric acid, 2-hydroxybutyric acid, 3-ureidopropionic acid, and proline. . The method according to any one of claims 6 to 8, for the diagnosis of a cat or dog at stage 1 or 2 of the IRIS diagnostic criteria for chronic kidney disease. A pharmaceutical or food composition containing one selected from the group consisting of taurine, proline, and γ-glutamyl ornithine, which is to be ingested by a subject with decreased renal function or a subject at risk thereof. 11. The composition according to claim 10, wherein the subject is a cat or a dog. 12. The composition according to claim 10, wherein the subject is a cat or dog in stage 1 or 2 of the IRIS diagnostic criteria for chronic kidney disease. The composition according to any one of claims 10 to 12, further comprising at least one selected from the group consisting of docosahexaenoic acid (DHA)-binding lipids and eicosapentaenoic acid (EPA)-binding lipids. The composition according to any one of claims 10 to 13, for ingestion by a subject diagnosed with renal function by the method according to any one of claims 1 to 9. Selected from the group consisting of docosahexaenoic acid (DHA)-bound lipids and eicosapentaenoic acid (EPA)-bound lipids for ingestion by a subject diagnosed with renal function by the method according to any one of claims 1 to 9. A pharmaceutical or food composition comprising at least one of: The composition according to any one of claims 13 to 15, comprising at least one selected from the group consisting of DHA-binding lipids and EPA-binding lipids as fish oil.

Images